Regulatory control of glutamate - induced neuronal superoxide production Project Summary/Abstract Glutamate excitotoxicity is a primary cause of cell death in stroke, brain trauma. Neuronal production of superoxide is necessary for excitotoxic cell death to occur. Glutamate-induced superoxide production has long been considered an inevitable, physical consequence of calcium influx and resulting mitochondrial dysfunction, but recent studies show that the superoxide is instead produced by the signaling enzyme, NADPH oxidase. Here we aim to delineate key regulatory steps in the signal transduction pathway linking neuronal glutamate receptor activation to NADPH oxidase activation, and the role of this process in local cell-to-cell propagation of excitotoxic injury. Preliminary studies suggest that key components of this pathway include the NR2B subunit of NMDA-type glutamate receptors, phosphoinositol-3-kinase (PI3K), PTEN, and phospholipase A2. The studies proposed here will evaluate the importance of each of these components using pharmacological, dominant negative, and genetic approaches. We also aim to identify mechanisms by which NADPH oxidase and mitochondria may interact in superoxide production. These studies will be performed using dissociated neuronal cultures, brain slices, and whole-animal experimental models. Successful completion of these studies will reconcile long-standing contradictions in this field, and will identify novel targets and mechanisms contributing to both acute and chronic neurological disorders. These studies will also further our understanding of the molecular framework for normal superoxide signaling between neurons, a process thought to modulate synaptic plasticity in brain. 1 PUBLIC HEALTH RELEVANCE: Glutamate - induced neuronal death is an important cause of neural injury in both acute-onset disorders such as stroke and trauma, and more chronic disorders such as amyotrophic lateral sclerosis and Alzheimer's disease. The reactive oxygen species, superoxide, is known to mediate glutamate-induced neuronal death. Studies proposed here will identify key steps leading from glutamate receptor activation to the production of superoxide, and thereby identify ways of blocking or modulating this important cell injury pathway.